基于关键酶PQQ-ADH代谢调控的果醋高强度发酵机理研究

Low productivity is the core issue that restricts the industrial production of fruit vinegar by liquid fermentation. Studies have shown that pyrroloquinoline quinine-dependent ethanol dehydrogenase (PQQ-ADH) plays an important role in promoting the ability of acetic acid production by Acetobacter. Therefore, it is a great significance to find the restriction mechanism of the key enzyme to the metabolic activity and regulate the metabolic activity of key enzyme by metabolism control. In the previous research, the adhA-adhB overexpression mutant has been successfully constructed, in order to explore the metabolic regulation function and the fermentation mechanism for high productivity，the applicants propose to carry out the following researches around the key enzyme PQQ-ADH: ① Studying the influence of the key enzyme on the metabolism network of Acetobacter: To state the functional mechanism of key enzyme on acetic acid production ability of Acetobacter, the fermentation characteristics and the substrate resistance ability of the mutant and the wild-type strain is contrastive analyzed scientifically; the changes of carbon flux, relevant enzymes activity and their gene expression level in the metabolic pathway, is also analyzed. ② Studying the metabolic regulation strategy of the key enzyme for fruit vinegar fermentation with high productivity: To obtain the semi-continuous fermentation system of fruit vinegar production with high productivity, the influnce of the key enzyme activator and the fermentation strategy on the ability of acid production are focused on; To reveal the mechanism for fruit vinegar fermentaiton with high productivity, the characteristics of the semi-continuous fermentation are studied, and the stress situation to the extreme environment of the relevant enzymes activities in metabolic network and the cell tissues are emphatically analyzed. The results are expected to provide certain reference for the fruit vinegar production by liquid fermentation with high productivity.

发酵强度低是制约液态发酵果醋工业化生产的核心问题。研究表明，依赖于吡咯喹啉醌的乙醇脱氢酶（PQQ-ADH）对醋酸菌的产酸能力具有重要的促进作用。因此，找出代谢活力关键酶的限制机制并开展深入调控具有重要意义。申请人在前期成功构建adhA-adhB过表达突变菌的基础上，拟围绕关键酶PQQ-ADH代谢调控功能及高强度发酵机制开展探索：①关键酶对细胞代谢网络的影响规律研究：对突变菌与原始菌的发酵特性及底物耐受性进行系统研究；分析代谢网络中碳流量、相关酶活力及其基因水平的变化态势，阐述关键酶对醋酸菌产酸能力的作用机制。②基于关键酶的果醋高强度发酵调控研究：重点开展关键酶激活剂及发酵策略的外源调控研究，形成果醋半连续高强度发酵体系；研究半连续发酵特性，着重分析代谢途径相关酶活力和细胞组织对环境聚变的响应，试图揭示果醋高强度发酵机理。研究结果期望为果醋的高强度液态发酵提供一定参考。

研究背景：果醋是目前高效液态纯种发酵代替传统酿制工艺的重要产品之一。主要由醋酸菌进行发酵获得。我国果醋加工起步较晚，口感和风味都不能满足要求。研究发现，吡咯喹啉醌乙醇脱氢酶（PQQ-ADH）对醋酸菌的细胞代谢和生产性能有重要的影响。基于PQQ-ADH的醋酸菌高效发酵代谢调控既可以初步明确PQQ-ADH功能特性，又可以实现果醋的高强度发酵。.主要研究内容：①关键酶PQQ-ADH对细胞代谢网络的影响规律研究：系统研究醋酸菌的发酵特性及耐受性，以及对乙醇、醋酸、温度的耐受性；研究代谢网络中碳代谢流量、相关酶活力及其基因表达水平的变化情况。②果醋半连续高强度发酵代谢调控研究：围绕提高、维持关键酶PQQ-ADH活力进行外源调控策略寻优；重点分析重复利用菌体半连续发酵果醋的代谢调控策略，揭示果醋半连续高强度发酵机理。.重要结果：本研究实施过程中，主要成果均是围绕PQQ-ADH对醋酸菌高强度发酵的作用，开展了菌株筛选、发酵特性、关键酶激活剂、半连续发酵关键参数探寻等工作，围绕这几个方面形成主要成果：获得优良突变菌1株，新专利5个（授权2个）、发表论文14篇（SCI、EI论文13篇、中文核心3篇）、培养研究生3名、出国访学1人。.关键数据：围绕果醋高强度发酵的代谢调控策略开展研究，有效提高了乙酸的产量，分析了发酵代谢通量，通过ADH的调控，有效提升乙酸通量，结合半连续发酵关键参数寻优，实现了高强度发酵果醋，半连续发酵第五批次40 h内，醋酸增产50 g/L，发酵强度达到了1.25 g/L•h。.科学意义：围绕乙醇脱氢酶开展强化醋酸菌氧化乙醇代谢途径的调控研究，对提高醋酸菌产酸能力、建立果醋高强度发酵体系具有重要意义。同时为解决液态发酵果醋风味不足、品质低问题，形成高品质果醋的液态发酵生产提供了有益参考。

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